Cardiogenic Gene Switch, Role of SRF Phosphorylation

描述（由申请人提供）：血清反应因子（SRF）作为主要的调控平台，由于其对中胚层形成的强制性要求以及其与其他早期心脏富集转录辅助因子形成特异性蛋白-蛋白关联的能力，可能在心脏祖细胞的承诺中发挥核心作用。对SRF无突变体的分析支持了SRF是哺乳动物心脏中胚层形成的重要调节因子的观点，并将SRF置于心脏细胞承诺和分化调节层次的非常高的位置。我们发现两个保守的SRF MADS盒残基Thr159和Ser162的磷酸化调控构成了SRF依赖性心源基因程序激活和抑制的新基因开关。蛋白激酶C （PKC）对SRF具有深远的抑制活性，其磷酸化主要指向Ser162，其次指向Thr159。VEGF可能通过Flkl受体发出信号并驱动PKC活性，部分通过阻断心源性收缩蛋白基因活性的表达来灭活SRF的肌生成活性。此外，VEGF可能在形成心脏形成区的后缘发挥抑制活性，并可能在心脏垫形成到无收缩心内膜出现的过渡过程中发出信号。磷酸化的SRF基因开关也可能以牺牲致心基因活性为代价，激活内皮血管生成程序。因此，本提案的中心主题将是确定SRF的磷酸化是如何在心脏中胚层的出现和胚胎小鼠心脏的发育和形成过程中通过网络调节下游基因靶标的，SRF位于MADS盒子中α - 1线圈中相距仅3个氨基酸的两个位点。目的一：SRF MADS box的磷酸化是否在小鼠心脏发生和发育过程中发生？目的II SRF MADS盒的磷酸化是否作为基因调控开关？目的III: SRF MADS box的磷酸化在确定早期心脏和/或内皮/血管生成程序中是否具有直接作用？目的四：VEGF信号通路是否抑制SRF依赖性心源性基因活性？

DESCRIPTION (provided by applicant): Serum response factor (SRF), as the master regulatory platform, may play a central role in the commitment of cardiac progenitors by virtue of its obligatory requirement for mesoderm formation and by its ability for making specific protein-protein associations with other early cardiac enriched transcription cofactors. Analysis of SRF null mutants supports the idea that SRF is an essential regulator of mammalian cardiac mesoderm formation and places SRF at a very high point in the regulatory hierarchy for cardiac cell commitment and differentiation. We discovered that the regulated phosphorylation of two conserved SRF MADS box residues Thr159 and Ser162 constitutes a novel gene switch for the activation and repression of SRF dependent cardiogenic gene programs. Protein kinase C (PKC) has a profound inhibitory activity on SRF, directing phosphorylation primarily to Ser162 and secondarily to Thr159. It is likely that VEGF by signaling through the Flkl receptor and driving PKC activity may inactivate SRF myogenic activity in part by blocking the expression of cardiogenic contractile protein gene activity. In addition, VEGF may exert inhibitory activity that forms the posterior border of the heart forming region and may signal later during the transition from forming the cardiac cushion to the appearance of the noncontractile endocardium. The phosphorylated SRF gene switch may also allow for the activation of the endothelial-hemangiogenic program at the expense of cardiogenic gene activity. Thus, the central theme of this proposal will be to determine how the phosphorylation of SRF, at two sites that are only three amino acids apart in the alpha 1 coil of the MADS box, is networked to regulate downstream gene targets during the appearance of cardiac mesoderm and the elaboration and formation of the embryonic mouse heart. By the following four aims: Aim I: Does phosphorylation of SRF MADS box occur during cardiogenesis and the elaboration of the murine heart? Aim II Does phosphorylation of the SRF MADS box act as a gene regulatory switch? Aim III Does phosphorylation of SRF MADS box have a direct role in specifying early cardiac and or endothelial/hemangiogenic programs? Aim IV Does the VEGF signaling pathway repress SRF dependent cardiogenic gene activity?